Conditional Protein Splicing Switch in Hyperthermophiles through an Intein-Extein Partnership

Area of Science:

• Biochemistry
• Molecular Biology
• Microbial Genetics

Background:

• Inteins are protein segments that excise themselves and ligate flanking exteins through autocatalysis.
• Some inteins regulate splicing based on environmental cues, representing a novel posttranslational control mechanism.
• RadA intein from *Pyrococcus horikoshii* requires temperature and single-stranded DNA (ssDNA) to overcome inhibitory intein-extein interactions.

Purpose of the Study:

• To investigate the temperature-dependent splicing of RadA intein from *Thermococcus sibericus*.
• To identify the structural elements responsible for regulating intein-extein interactions and splicing activity.
• To elucidate the role of the nonconserved region (NCR) in controlling protein splicing in response to environmental stimuli.

Main Methods:

• Comparative analysis of RadA inteins from *T. sibericus* and *P. horikoshii*.
• Site-directed mutagenesis, including deletion of the nonconserved region (NCR).
• In vitro protein splicing assays at varying temperatures and in the presence of ssDNA.

Main Results:

• *T. sibericus* RadA splicing is activated at lower temperatures than *P. horikoshii* RadA.
• The nonconserved region (NCR) of the intein is critical for efficient and accurate splicing with native exteins.
• Deletion of the NCR significantly impairs splicing, but this effect is partially rescued by ssDNA, indicating the NCR acts as a regulatory switch.

Conclusions:

• The NCR functions as a flexible hinge, controlling long-range intein-extein interactions that inhibit splicing.
• Environmental signals like temperature and ssDNA modulate the NCR's conformation to facilitate active site assembly.
• Inteins have evolved beyond self-excision to actively regulate host protein function, a potentially widespread mechanism in microbes.